1. Field of the Invention
The present application relates to microparticles, in particular silicon microparticles, and a method for coating these.
2. Description of Related Art
Several publications (for example European Patent EP 1276555B1) describe the fabrication of microparticles, notably silicon microparticles, using wafer based microfabrication techniques. Biological probes, such as antibodies or nucleic acids, can then be attached to those microparticles using techniques known in the art and the microparticles can then subsequently be used for the detection of target analytes in a sample.
However, when attaching fluorescent probes (typically ssDNA probes labelled with FAM fluorophore at the 5′ end) on the surface of silicon microparticles and performing subsequently hybridization assays with complementary targets labelled with Cy5 fluorophore did not reveal any fluorescence signal neither on the FAM channel before hybridization or on the Cy5 channel after hybridization.
After investigation, this appeared to be caused by destructive light interference occurring right at the surface of the partially reflective silicon microparticles. This phenomenon is described in several scientific publications (Bras, M., et al., Optimisation of a silicon/silicon dioxide substrate for a fluorescence DNA microarray. Biosensors & bioelectronics, 2004. 20(4): p. 797-806; Voile, J. N., et al., Enhanced sensitivity detection of protein immobilization by fluorescent interference on oxidized silicon. Biosensors and Bioelectronics, 2003. 19(5): p. 457-464). The same phenomenon would be observed to some extent on any material that is reflective.
The scientific publications mentioned above suggest the addition of an oxide layer at optimized thickness to move the fluorophores away from the surface and reverse this effect into a constructive light interference which reveals and potentially amplifies the fluorescence signal. A similar method, i.e. the addition of a transparent layer (silicon dioxide, silicon nitride, or others) with the proper thickness, could be applied to other materials than silicon as a substrate.
The reflection coefficient of a polished silicon wafer (or SOI (silicon-on-insulator) wafer) is about 35-40% in the 500-700 nm wavelength range. This means that significant light interference occurs in this range, which includes the common fluorescent labels such as FAM, Cy3, and Cy5. The interference effect is dependent on the thickness of the layer (oxide in this case) that separates the fluorescent labels from the reflective surface of the silicon. In particular cases the interference can completely anneal the fluorescent signal while it can strongly enhance the signal in other cases. The interference can be either destructive or constructive, as function of the thickness of the layer, of the refractive index of the layer, of the wavelength of the light, and of its angle of incidence. For instance, at quasi normal incidence, constructive light interference of a light beam reflected specularly by a substrate is maximal at the position of the electromagnetic field “anti-nodes” which are located periodically at distances dan=(2k+1)λeff/4 from the substrate; k is a positive integer and λeff is the wavelength of light in the propagation medium. Thus, for k=0, dan=λeff=λcy5/noxide/4=654/1.45/4=113 nm for the common Cy5 fluorescent label, where λcy5 is the mean value between the absorbance peak and the emission peak and where noxide is the refractive index of silicon dioxide. Thus, in order to achieve maximal constructive light interference, the layer needs to have a thickness of ca. 113 nm. The same reasoning applies to any other fluorescent label.
The scientific publications referenced above describe methods to add an oxide layer on top of the silicon substrates. However, those methods are only applicable to large substrates that can be handled easily and that are already in their final format. For small microparticles in the micron range, typically from 1 to 300 micron, such as those that are contemplated herein, these methods are not readily applicable. Typically, such small microparticles are handled “in batch” as a powder, in suspension or temporarily tethered to larger substrates and the methods disclosed in the art do not teach how to perform the required steps in such conditions.
The fabrication of microparticles by using wafer-based microfabrication techniques is well known in the art. These techniques typically make use of particular wafers (SOI wafers) that have an insulator layer. The microparticles can be structured in the top device layer and then released by etching away the buried insulator layer. For example, WO 2009/014848, WO 07/081410, GB2306484, US2003/203390, EP 1 018 365 and EP 2 113 301 describe the fabrication of various types of (generally encoded) microparticles with various shapes and structures.
However, there is a need in the art for fabrication techniques providing for the addition of an essentially transparent layer that would contribute to the construction of a positive interference as described above. The problem of adding a transparent layer is particularly challenging when the material constituting this transparent layer is the same as the material used as the insulator layer (buried insulator layer in SOI wafer) because such additional layer would be partially or totally removed when etching away the insulator, typically in solution or in vapor of HF (hydrofluoric acid solution).
A possible way to overcome this problem and to perform the coating of those microparticles could be the use of conventional suspension techniques. Yet, there is no known method that would allow achieving a coating with a layer that has the required thickness and the required regularity/repeatability. Indeed, an irregular or a different thickness at each batch would result in irregular or different fluorescent signals readouts at each time, for the same amount of attached fluorophores (i.e. the same amount of labeled targets being captured), which renders quantization difficult or impossible. For example, a thin oxide layer of a few nanometers, typically 3 nm, as obtained when oxidizing the microparticles in piranha solution (mixture of sulfuric acid H2S04 and hydrogen peroxide H202 in typical ratio of 3:1), would prevent any fluorescence readout perpendicular to the microparticles by producing destructive light interference. Another example is the formation of a thin silica (silicon dioxide) shell around the microparticles using a sol-gel method similar to silica coating of nanoparticles (Graf C, Vossen D U, Imhof A, van Blaaderen A. A General Method To Coat Colloidal Particles with Silica. Langmuir. 2003; 19(17):6693-6700) but does not provide a controlled and homogeneous layer and does not achieve required thickness of approx. 100 nm. The sol-gel process, also known as chemical solution deposition, is a wet-chemical technique widely used in the fields of materials science used primarily for the fabrication of materials (typically an oxide) starting from a chemical solution (or sol) that acts as the precursor for an integrated network (or gel) of either discrete particles or network polymers.
Polymer layers instead of an oxide layer may also be considered but, here again, it becomes difficult to get layers with the required thickness (above 100 nm) and homogeneity.
There is therefore a need in the art for a method that allows the production of microparticles with a layer, preferably a transparent layer, more preferably an oxide layer, on their surface having the required thickness and regularity/repeatability.